A Sister Chromatid Cohesion Assay Using Xenopus Egg Extracts

2016 ◽  
pp. 3-21 ◽  
Author(s):  
Keishi Shintomi ◽  
Tatsuya Hirano
Keyword(s):  
Sister Chromatid ◽  
Sister Chromatid Cohesion ◽  
Chromatid Cohesion ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

scholarly journals Identification and Characterization of Sa/Scc3p Subunits in the Xenopus and Human Cohesin Complexes

2000 ◽  
Vol 150 (3) ◽  
pp. 405-416 ◽  
Author(s):  
Ana Losada ◽  
Tomoki Yokochi ◽  
Ryuji Kobayashi ◽  
Tatsuya Hirano
Keyword(s):  
Sister Chromatid ◽  
Somatic Tissue ◽  
Sister Chromatid Cohesion ◽  
Dominant Form ◽  
Culture Cells ◽  
Chromatid Cohesion ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

A multisubunit protein complex, termed cohesin, plays an essential role in sister chromatid cohesion in yeast and in Xenopus laevis cell-free extracts. We report here that two distinct cohesin complexes exist in Xenopus egg extracts. A 14S complex (x-cohesinSA1) contains XSMC1, XSMC3, XRAD21, and a newly identified subunit, XSA1. In a second 12.5S complex (x-cohesinSA2), XSMC1, XSMC3, and XRAD21 associate with a different subunit, XSA2. Both XSA1 and XSA2 belong to the SA family of mammalian proteins and exhibit similarity to Scc3p, a recently identified component of yeast cohesin. In Xenopus egg extracts, x-cohesinSA1 is predominant, whereas x-cohesinSA2 constitutes only a very minor population. Human cells have a similar pair of cohesin complexes, but the SA2-type is the dominant form in somatic tissue culture cells. Immunolocalization experiments suggest that chromatin association of cohesinSA1 and cohesinSA2 may be differentially regulated. Dissociation of x-cohesinSA1 from chromatin correlates with phosphorylation of XSA1 in the cell-free extracts. Purified cdc2-cyclin B can phosphorylate XSA1 in vitro and reduce the ability of x-cohesinSA1 to bind to DNA or chromatin. These results shed light on the mechanism by which sister chromatid cohesion is partially dissolved in early mitosis, far before the onset of anaphase, in vertebrate cells.

scholarly journals ISWI Remodeling Complexes in Xenopus Egg Extracts: Identification as Major Chromosomal Components that Are Regulated by INCENP-aurora B

2002 ◽  
Vol 13 (1) ◽  
pp. 25-39 ◽  
Author(s):  
David E. MacCallum ◽  
Ana Losada ◽  
Ryuji Kobayashi ◽  
Tatsuya Hirano
Keyword(s):  
Chromosome Condensation ◽  
Aurora B ◽  
Mitotic Chromosomes ◽  
Novel Proteins ◽  
Condensin Complex ◽  
H3 Phosphorylation ◽  
Chromatid Cohesion ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

We previously characterized major components of mitotic chromosomes assembled in Xenopus laevis egg extracts and collectively referred to them as Xenopuschromosome–associated polypeptides (XCAPs). They included five subunits of the condensin complex essential for chromosome condensation. In an effort to identify novel proteins involved in this process, we have isolated XCAP-F and found it to be theXenopus ortholog of ISWI, a chromatin remodeling ATPase. ISWI exists in two major complexes in Xenopus egg extracts. The first complex contains ACF1 and two low-molecular-weight subunits, most likely corresponding to Xenopus CHRAC. The second complex is a novel one that contains theXenopus ortholog of the human Williams syndrome transcription factor (WSTF). In the absence of the ISWI complexes, the deposition of histones onto DNA is apparently normal, but the spacing of nucleosomes is greatly disturbed. Despite the poor spacing of nucleosomes, ISWI depletion has little effect on DNA replication, chromosome condensation or sister chromatid cohesion in the cell-free extracts. The association of ISWI with chromatin is cell cycle regulated and is under the control of the INCENP-aurora B kinase complex that phosphorylates histone H3 during mitosis. Apparently contradictory to the generally accepted model, we find that neither chromosome condensation nor chromosomal targeting of condensin is compromised when H3 phosphorylation is drastically reduced by depletion of INCENP-aurora B.

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